Autoflex 2 tof tof

MALDI-TOF/TOF MS/MS was performed with an Autoflex TOF/TOF II (Bruker Daltonics Ltd.) fitted with a nitrogen laser operating at 337 nm. Full-scale MS scan negative ions were analyzed in reflectron mode with norharmane (10 mg/ml in water plus 0.1% trifluoroacetic acid) as the matrix. The instrument was externally mass calibrated. For MS/MS, negative precursor ions were analyzed by LIFT and data were recorded with flexControl version 3.0 and processed with flexAnalysis version 3.0 at Durham University (Mass Spectrometry Service, Department of Chemistry, Durham University, United Kingdom).

One microliter of a 10 mg/mL solution of 2,5-dihydroxybenzoic acid (DHB) in 33% ethanol was applied to a MTP 384 ground steel TF target plate (Bruker Daltonics). The 1 µL sample prepared above was then mixed into the DHB droplet and dried under a stream of air. The samples were analyzed with an Autoflex TOF/TOFII instrument (Bruker) with a nitrogen 377 nm laser beam. The instrument was operated in positive acquisition mode and controlled using the FlexControl 2.2 software package. All of the spectra were obtained using the reflector mode with an acceleration voltage of 19 kV, a reflector voltage of 20 kV, and pulsed ion extraction of 140 ns in the positive ion mode over an acquisition range (m/z) of 600–7000. The data were collected from an average of 500 laser shots, using the lowest laser energy necessary to obtain sufficient signal to noise ratios. Peak lists were generated from the MS spectra using the Bruker Flex Analysis software (Version 3.0). Post-source decay spectra using the Bruker Daltonics LIFT system were recorded at precursor ion acceleration and fragment acceleration voltage of 18.96 kV (LIFT voltage 4.37 kV). The reflector voltages 1 and 2 were set to 23.49 and 9.69 kV, respectively. The samples were also analyzed by no-matrix MALDI-TOF-MS over an acquisition range (m/z) of 100–2000 in positive or negative ion mode.^{45 (link)}

2,5‐Dihydroxyactetophenone (2,5‐DHAP, Bruker Daltonik) was used as matrix for MALDI‐TOF MS. For solubilisation of the matrix, 7.6 mg of 2,5‐DHAP were dissolved in 375 μL of absolute ethanol. After this, 125 μL of an 18 mg mL⁻¹ aqueous solution of diammonium hydrogen citrate (Sigma–Aldrich) were added.
Protein samples were desalted by chloroform/methanol protein precipitation as described elsewhere.29 The dried protein pellets were dissolved in 50–100 μL 0.1 % TFA solution. A 2 μL aliquot of this desalted protein sample was mixed with 2 μL of 2 % TFA solution. After addition of 2 μL of matrix solution, the mixture was pipetted up and down until the crystallisation started and the solution became cloudy. Finally, 0.5 μL of the crystal suspension was spotted onto the ground steel target plate and the droplet was air‐dried completely at room temperature.
Spectra were acquired with an autoflex II TOF/TOF (Bruker Daltonik) in positive linear mode in combination with the flexControl software (Version 3.3, Bruker Daltonik) and analysed with the flexAnalysis software (Version 3.3, Bruker Daltonik). Theoretical molecular weights were calculated using the Compute pI/Mw tool on the ExPASy Server.